Two biologists, split by rivalry and disagreement, suddenly realised that they were on the same side. The result was a revolutionary breakthrough. Special report by our Science Editor, Steve Connor

Steve Connor - Saturday 07 April 2012

A dogma has haunted the study and treatment of female infertility for more than half a century. It states that a baby girl is born with an ever-diminishing number of egg cells which cannot be renewed or replenished during her life, and that when she runs out of these eggs an irreversible menopause begins.

The dogma's origins go back to 1951 when Sir Solly Zuckerman, a South African born British scientist, published the definitive study showing “unequivocally” that the human ovary, like that of other mammals, has a finite resource of egg cells that begins to be lost during foetal development, even before a woman is born.

Estimates suggested that the female foetus has about 7 million putative egg cells that reduce to about 1 million at the point of birth. By the time of puberty, the teenage girl has between about 300,000 and 400,000 egg cells and these are lost at a rate of about 1,000 a month, with typically just one ripening to maturity at the time of ovulation.

Menopause usually begins, so the dogma goes, in late middle age when the egg bank runs out of its most valuable currency. It is then, for many women, that age-associated health problems begin, from hot flushes and osteoporosis to heart disease and cognitive decline.

The problem with the dogma, and Zuckerman's “definitive” study, was that it turns out not to be true. In a series of remarkable studies published over the past eight years scientists have produced convincing evidence to suggest that women are not after all born with all the egg cells that they will ever possess.

Several research teams across the world have shown that the mammalian ovary is far more versatile than Zuckerman and his subsequent followers had ever thought possible. The core of this new thinking lies in the remarkable discovery of “oogonial stem cells” within the ovary that are capable of producing a constant supply of fresh eggs, or oocytes.

The discovery of these stem cells, which had gone unrecognised for six decades, has profound implications for the study and treatment of female fertility, as well as a plethora of other health conditions.

It raises the prospect of being able to grow unlimited numbers of human oocytes in the laboratory that could either be used in IVF treatment or as a source of embryonic stem cells to treat incurable illnesses such as Parkinson‘s disease.

But, even more profound, is the idea that human ovaries might be induced in some way to function well into old age, not for the sake of maintaining fertility but to retain the other health benefits that stem from active, egg-filled ovaries.

Some scientists are seriously suggesting that this work will lead to reversing or at least delaying the menopause, and a few even talk of an “elixir of youth” that will allow women to regain the good health they enjoyed when they were young and fertile.

“To me, there is a grander golden chalice here which is ageing itself. These cells may provide a way for us to tackle that tremendously important problem,” said Professor Jonathan Tilly of Harvard Medical School in Boston, Massachusetts.

“It's very clear that keeping the ovaries working has tremendous health benefits on the ageing female body,” Professor Tilly said.

It was Tilly's pioneering work in 2004 that kick-started the revolution now sweeping through fertility laboratories around the world. It was he who discovered and named the oogonial stem cells in the mammalian ovary.

Like so many important scientific discoveries, this one was unexpected and serendipitous. Tilly was working on a quite different problem when he and his team discovered something that they could not explain.

“We never set out to disprove the dogma. In fact we were looking at cell death, to better define why egg cells die and how they die. Ten years ago, like everyone else, I believed the dogma was correct,” Professor Tilly said.

He had developed a technique for counting the rate at which ovary cells die and the findings were puzzling. To summarise, he found that cells were dying about three times faster than they should be, which meant that the egg cell deposit should be running out even faster than it does - something must be replacing them.

“When we first saw this we were in complete shock and disbelief because we, like everyone else, believed the dogma, but yet we had a mathematical dilemma that told us otherwise,” he said.

“I was in such shock that we did it over and over again, but we came to the same conclusion each time. We were completely caught off guard.”

When Tilly published his study in the journal Nature in 2004 the fertility community was not pleased. Understandably, he was subjected to rigorous scientific scrutiny that continued for many years afterwards, but some comments were designed to be deliberately cruel.

“It was discouraging to hear so many people voice negative comments about the work, and many of the comments were not based on science but were personal opinions and beliefs,” Tilly said.

The work was carried out on mouse ovaries, the standard animal model, and some critics suggested the findings may be an artefact of murine tissue. One reviewer helpfully suggested that humans are not big mice, unless you live in Disneyland.

Evelyn Telfer of Edinburgh University was one of Tilly’s kinder critics. She said she just couldn’t bring herself to believe something that broke the Zuckerman rule on which she was brought up.

“Everything we did was informed by this dogma. Everything was based on the idea that there could be no more new oocytes. We approached everything on the basis of a fixed population of egg cells,” Dr Telfer said.

“Tilley was relatively new to the field when he published that paper in 2004 and other people already in the field said that there must be other, alternative explanations for his results,” she said.

“The problem at that time was that it [Tilly’s 2004 study] wasn’t the cleanest of experiments to absolutely show the existence of oocyte stem cells,“ she added.

“It was very bold of Tilley because he was reporting results that went against the dogma. It would have been so easy for him to try to fit them into the dogma. It made people sit up and think.”

The real problem was trying to isolate and characterise these supposed stem cells. Tilly wrongly suggested in 2005 that they could reside in the bone marrow and migrate to the ovaries.

Then a key breakthrough happened in 2009 when a Chinese group led by Ji Wu of Shanghai Jiao Tong University published a study claiming to have isolated stem cells in mouse ovaries which Wu had used to generate mature egg cells. She reported the successfully fertilisation of these eggs, resulting in offspring.

Again, many remained sceptical, but Telfer was no longer one of them: ”The breakthrough for me in my relationship with Tilley was when the Chinese group published their paper in 2009.“ She was a convert to the dogma-breaking minority.

Tilly began furious attempts to replicate Wu’s work but, like many others trying to do the same, it defied repetition - which is the key to science. Wu’s description of her methodology appeared incomplete.

“We tried for nine months to get her protocol to work. If I was a sceptic of this work I would have tried three times and quit and say that it's not repeatable,” Professor Tilly said.

“But we spent months and months working with the protocol to get it to work. It was nine months of non-stop trial and error before we succeeded and I still don’t know what we did right because we had changed and tinkered with so many different things,” he said.

Armed with this new protocol, Tilly was about to make his most important breakthrough.

While attending a scientific conference in Tokyo he met up with a former colleague, Yasushi Takai of Saitama Medical University, who had a bank of frozen human ovaries taken from Japanese women undergoing sex-change treatment.

Here was the opportunity for Tilly to try out his new protocol for isolating ovarian stem cells on genuine human tissue. He lost no time in showing that these elusive cells do indeed exist in humans and that they could be induced to develop into very early stage oocytes - he did not, after all, live in Disneyland.

“It worked the first time. The human cells popped out and they had every attribute the mouse cells had,” Professor Tilly said.

When he inserted the human oogonial stem cells into mouse ovarian tissue he witnessed their partial development down the path towards fully mature oocytes, but only partial.

What was needed was some way of bringing these very early eggs to full development. It was only by ripening them completely and allowing them to be fertilised by a human sperm that he could finally prove the existence of true oogonial stem cells in the human ovary.

Tilly then remembered his one-time critic in Edinburgh. Telfer had pioneered a technique for bringing egg cells to full maturity from the precise developmental stage that Tilly was able to reach with his oogonial stem cells.

“There is a common juncture point where we can get to and from where she starts. Sticking the two procedures together was a no-brainer,” Professor Tilly said.

The Edinburgh University team has already received material and expertise from Tilly and Dr Telfer is now about to apply for a licence from the Human Fertilisation and Embryology Authority (HFEA) to carry out the first fertilisation of a mature human egg derived from oogonial stem cells grown in a laboratory dish.

If an early human embryo is produced in this way it will dramatically alter the landscape for human fertility treatment. Women and girls undergoing ovary-destroying chemotherapy or radiation treatment may be able in the future to have their oogonial stem cells stored and banked so that they could later have an unlimited supply of their own eggs for IVF treatment.

It will also mean that science has a way of producing egg cells in the laboratory for a range of experimental and practical purposes. One of these could lead to a fundamental understanding of the menopause itself - why do women, unlike men, stop producing their sex cells in middle age?

Answering this question could be the key to solving many of the health problems associated with female ageing. Tilly and Telfer both believe that the menopause may not be due to a shortage of egg cells per se, but a depletion of the cells within the ovary that are needed to support and nurture oocytes.

If this is the case, and if Tilly’s oogonial stem cells can be used to replenish human egg cells or used to develop ways of extending the life of their vital support cells in the human ovary, the menopause could indeed be delayed or reversed.

“Personally I don't think it's hype because that's our goal. The mouse studies show it can be done, that you can essentially delay the menopause to very advanced ages simply by maintaining an adequate reserve of egg cells in the ovaries,” Professor Tilly said.

And if it is possible to delay the menopause, there will hopefully be associated health benefits for older women. But could it really be an elixir of youth?

“From the mouse studies, if you keep the ovaries working, yes it's an elixir of youth without any increased risk of cancer. It's published and it's indisputable,” Professor Tilly said.

Case Study:

An Egg Donor

"Women are put off donating eggs by how painful and arduous the process is"

Sylvia Barr, from south London, was 34 when she donated an egg in 1991. She said she felt the urge to do so after conceiving her own son Eliott thanks to a sperm donor.

"In 1990, I was 33 and I decided I wanted to be a mother. I didn’t have a partner, I had been through some unsuccessful relationships so I decided I wanted to have a child on my own. I had IVF treatment with donated sperm and I was lucky enough that the procedure worked first time.

“I felt that I had been helped by an altruistic stranger and I asked myself what I could do to help someone else. I didn’t want any more children beyond my son for financial reasons. I believe that, in life, you don’t just take; you have to give back as well. A stranger helped me have a child, I wanted to reciprocate.

“There is still not an abundance of egg donors. I think people are put off by how hard and time-consuming the process is. A lot of people don’t understand what is involved: I had to give myself injections, use the nasal spray and go under an anaesthetic.

“But it was worth it in the end. It was something I wanted to do, to give something back. It was a way of evening things out for me, it was very important.

“I was driven by my own circumstances and seeing my own son made me want to help someone else find the same happiness. I didn’t mind who got my egg but if I could help them have that same joy, then I was happy to do it.

“I knew the process was painful and arduous, I had counselling to explain the implications and get me through the treatment. Your own health is put at risk; you are put on synthetic drugs and given hormones.

“You can produce too many eggs, making your ovaries enlarged, which is a serious condition, and I was told there is a higher risk of cancer. But the main issue is psychological. Understanding and being prepared to donate potentially human life is a big thing to deal with.

“I never worried about scarcity of embryos in my later life because you produce many more than you ever need. The ovary is full of potential life, so I didn’t think of it is a problem. But I have read since that there is an increased risk of infertility in people who have donated eggs."

Case Study:

Early menopause

"It was like my sister was just lending me her make-up or something"

Claire Cousins, 27, from Oxfordshire, had tried unsuccessfully to have a child for three years. After she found out she had gone through the menopause early at the age of 22, her sister agreed to donate an egg.

"I felt funny when I thought of having an unknown donor’s egg. I wanted my genes in my baby. My initial thought was that it was a three-year waiting list. Before looking into what the whole process actually meant, my friends said they would give me an egg.

"The way my sister looked at it was that her family was complete. Obviously, there is always the chance that the procedure could ruin her own fertility but she thought: ‘I am not using the egg, so go ahead’. It was like she was just lending me her makeup or something.

"I have spoken to some friends who said they are not sure that they could do it, but my sister’s attitude seemed to be: 'if that’s what you need, then that’s what I will give you’. Harry is 13-months-old now. Receiving that egg was the best thing in the world; I cherish him every day.

"We had to go through counselling individually and as a group with my sister’s husband. I had to have scans done on my womb but it was my sister who had to go through the worst. She had to have injections and use a nasal spray.

"I can understand why people don’t want to do it, it is a long process and it is harder for a woman to donate an egg than it is for a man to donate sperm. As a woman, you have to have an operation and be put under anaesthetic. I can understand it’s hard, but if it means that someone can have a baby, then as many people as possible should do it.

"You have to be strong enough to understand that someone else is going to have a baby and that the baby is not yours; you need to be prepared mentally.

"There is an issue in potential donors’ minds, of the risks to their own fertility. If you feel you are going to try for a baby again later in your life, then maybe you are not mentally ready. The best situation is if you think your family is complete.

"Hospitals advise you to think in those terms and you have to be at a certain age before you can donate to be on the safe side and you are told that the process could affect your own fertility."